[Capabilities of using a multifunctional led device in the study of objects of forensic examination]. / Vozmozhnosti ispol'zovaniya mnogofunktsional'nogo svetodiodnogo pribora v issledovanii ob"ektov sudebnykh ekspertiz.

The purpose of the work is to determine the capabilities of the modern multifunctional device «Photobox 3138¼ with LED illuminators with a high color rendering index for photofixation, colorimetric, spectrozonal and multispectral analyzes of forensic objects. The competencies of the «Photobox 3138¼ device were experimentally revealed in terms of visualization and photofixation of both traces of biological origin (blood, sweat fat, saliva, etc.) and traces of traditional examinations, including shot products, oil products. The design of the device with a working field of 300×300 mm provides for the optimal arrangement of LED illuminators, including white light with a high color rendering index; spectrum-zonal illuminator with 4 types of LEDs with narrow non-intersecting spectral lines (458.1; 523.1; 594.1 and 630.6 nm) in the visible range of the spectrum; UV- (370 nm) and IR- (850 nm) illuminators. The fundamental possibility of using the resulting digital images of forensic objects for subsequent mathematical processing is experimentally shown. Images taken in different spectral ranges help to detect traces and damage. It was found that «Photobox 3138¼ allows you to solve a variety of diagnostic tasks related to the search, visualization, fixation and analysis of trace information.

Life time of some RNA products of rDNA intergenic spacer in HeLa cells.

In human cells, the intergenic spacers (IGS), which separate ribosomal genes, are complex approximately 30 kb-long loci. Recent studies indicate that all, or almost all, parts of IGS may be transcribed, and that at least some of them are involved in the regulation of the ribosomal DNA (rDNA) transcription, maintenance of the nucleolar architecture, and response of the cell nucleus to stress. However, since each cell contains hundreds not quite identical copies of IGS, the structure and functions of this locus remain poorly understood, and the dynamics of its products has not been specially studied. In this work, we used quantitative PCR to measure the expression levels of various rDNA regions at different times after inhibition of the transcription by Actinomycin D applied in high doses. This approach allowed us to measure real or extrapolated half-life times of some IGS loci. Our study reveals characteristic dynamic patterns suggestive of various pathways of RNA utilization and decay.

Fluctuations of pol I and fibrillarin contents of the nucleoli.

Nucleoli are formed on the basis of ribosomal DNA (rDNA) clusters called Nucleolus Organizer Regions (NORs). Each NOR contains multiple genes coding for RNAs of the ribosomal particles. The prominent components of the nucleolar ultrastructure, fibrillar centers (FC) and dense fibrillar components (DFC), together compose FC/DFC units. These units are centers of rDNA transcription by RNA polymerase I (pol I), as well as the early processing events, in which an essential role belongs to fibrillarin. Each FC/DFC unit probably corresponds to a single transcriptionally active gene. In this work, we transfected human-derived cells with GFP-RPA43 (subunit of pol I) and RFP-fibrillarin. Following changes of the fluorescent signals in individual FC/DFC units, we found two kinds of kinetics: 1) the rapid fluctuations with periods of 2-3 min, when the pol I and fibrillarin signals oscillated in anti-phase manner, and the intensities of pol I in the neighboring FC/DFC units did not correlate. 2) fluctuations with periods of 10 to 60 min, in which pol I and fibrillarin signals measured in the same unit did not correlate, but pol I signals in the units belonging to different nucleoli were synchronized. Our data indicate that a complex pulsing activity of transcription as well as early processing is common for ribosomal genes.

Nucleolar DNA: the host and the guests.

Nucleoli are formed on the basis of ribosomal genes coding for RNAs of ribosomal particles, but also include a great variety of other DNA regions. In this article, we discuss the characteristics of ribosomal DNA: the structure of the rDNA locus, complex organization and functions of the intergenic spacer, multiplicity of gene copies in one cell, selective silencing of genes and whole gene clusters, relation to components of nucleolar ultrastructure, specific problems associated with replication. We also review current data on the role of non-ribosomal DNA in the organization and function of nucleoli. Finally, we discuss probable causes preventing efficient visualization of DNA in nucleoli.

Proliferative and inflammatory factors in the vitreous of patients with proliferative diabetic retinopathy.

PURPOSE: The purpose was to measure the concentrations of various cytokines and growth factors (including vascular endothelial growth factor [VEGF] and pigment epithelium-derived factor [PEDF]) in the vitreous of patients with proliferative diabetic retinopathy (PDR) and to investigate interaction between inflammatory and proliferative factors in the genesis of PDR. Materials and Methods : Vitreous samples from 32 eyes with PDR and 25 eyes without diabetes mellitus and signs of DR (control) were collected. Vitreous concentrations of VEGF, PEDF, monocyte chemotactic protein-1 (MCP-1), interleukin-4 (IL-4), IL-6, IL-8, IL-10, IL-17A, and secretory immunoglobulin A (sIgA) were simultaneously measured using enzyme-linked immunoassay. Results : Vitreous levels of VEGF, PEDF, IL-17A, IL-6, IL-8, IL-4, and sIgA were significantly (Π < 0.05) higher in eyes with PDR compared to control. The concentration of VEGF was more than 17-times higher than in control, and the concentration of PEDF was not changed oppositely and was also higher (1.45-times) compared to control, that may indicate disturbances of compensatory mechanisms in angiogenesis regulation in PDR. Significant (Π < 0.05) positive correlations were observed between vitreous concentrations of VEGF and IL-17ΐ (r = 0.45), VEGF and IL-8 (r = 0.48), VEGF and IL-4 (r = 0.51), PEDF and IL-17ΐ (r = 0.48), PEDF and IL-8 (r = 0.59), MCP-1 and PEDF (r = 0.72), MCP-1 and IL-8 (r0 = 0.45), IL-4 and IL-17ΐ (r = 0.65), IL-4 and IL-8 (r = 0.71), IL-8 and IL-17ΐ (r = 0.59). CONCLUSIONS: Significantly raised levels of inflammatory and proliferative factors and numerous positive correlations between them may demonstrate a significant role of activation of vascular proliferation and local inflammation in the pathogenesis of PDR.

Separation of replication and transcription domains in nucleoli.

In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.

[Evidence-based treatment of combined rotator cuff and SLAP lesions]. / Evidenzbasierte Behandlung der kombinierten Rotatorenmanschetten- und SLAP-Läsion.

PURPOSE: In the face of improved radiological and arthroscopic techniques the diagnosis and treatment of SLAP lesions has recently gained much interest. Originally described as an (isolated) injury of the overhead athlete, it was only recently that the association of SLAP and rotator cuff defects was described in up to 40 % of cases. This study addresses the question of the evidence-based treatment of such frequent, combined lesions. METHODS: Based on a systematic review of the online databases PubMed, EMBASE, CINAHL and Cochrane Library we identified clinical studies on the treatment of combined SLAP and rotator cuff lesions. Study quality was assessed using levels of evidence and a modified Jadad score. Clinical outcome was assessed through scores and range of motion assessments. RESULTS: We included 7 studies of 374 patients with a mean age of 53 ± 11 years followed for 35 ± 13 months. Combined lesions have a significant negative effect on isolated rotator cuff or SLAP repair. Patients older than 45 years of age had a significantly better clinical result after biceps tenotomy than SLAP repair with concomitant rotator cuff repair. Biceps tenotomy plus rotator cuff repair showed significantly better range of motion for flexion and rotation than SLAP plus rotator cuff repair. CONCLUSION: The frequent combination of SLAP and rotator cuff injury should be considered during assessment and informed consent of shoulder patients. While young patients and isolated SLAP lesions show excellent clinical results after elective repair, combined lesions should be treated with biceps tenotomy and/or debridement plus rotator cuff repair in patients older than 45 years.

Positioning of the NOR-bearing chromosomes in relation to nucleoli in daughter cells after mitosis.

It is known that chromosomes occupy non-random positions in the cell nucleus. However, it is not clear to what extent their nuclear positions, together with their neighborhood, are conserved in daughter cells. To address specific aspects of this problem, we used the model of the chromosomes carrying ribosomal genes that are organized in clusters termed Nucleolus Organizer Regions (NORs). We compared the association of chosen NOR-bearing chromosomes (NOR-chromosomes) with nucleoli, as well as the numbers of nucleoli, in the pairs of daughter cells, and established how frequently the daughter cells had equal numbers of the homologs of certain NOR-chromosomes associated with individual nucleoli. The daughter cells typically had different numbers of nucleoli. At the same time, using immuno-FISH with probes for chromosomes 14 and 15 in HeLa cells, we found that the cell pairs with identical combinations appeared significantly more frequently than predicted by the random model. Thus, although the total number of chromosomes associated with nucleoli is variable, our data indicate that the position of the NOR-bearing chromosomes in relation to nucleoli is partly conserved through mitosis.

NORs and their transcription competence during the cell cycle.

In human cells ribosomal genes are organized as clusters, NORs, situated on the short arms of acrocentric chromosomes. It was found that essential components of the RNA polymerase I transcription machinery, including UBF, can be detected on some NORs, termed "competent" NORs, during mitosis. The competent NORs are believed to be transcriptionally active during interphase. However, since individual NORs were not observed in the cell nucleus, their interphase status remains unclear. To address this problem, we detected the competent NORs by two commonly used methods, UBF immunofluorescence and silver staining, and combined them with FISH for visualization of rDNA and/or specific chromosomes. We found that the numbers of competent NORs on specific chromosomes were largely conserved in the subsequent cell cycles, with certain NOR-bearing homologues displaying a very stable pattern of competence. Importantly, those and only those NORs that were loaded with UBF incorporated bromo-uridine in metaphase after stimulation with roscovitine and in telophase, suggesting that competent and only competent NORs contain ribosomal genes transcriptionally active during interphase. Applying premature chromosome condensation with calyculin A, we visualized individual NORs in interphase cells, and found the same pattern of competence as observed in the mitotic chromosomes.

[Argyrophilic proteins in the regions of nucleolar organizers are markers of cell proliferation rate].

The paper reviews the literature on the role of the above proteins in the mechanisms of cell cycle regulation and presents data on changes in protein expression in dynamics of cell cycle. The significance of studies dealing with the expression of these proteins, which, along with the assessment of a cell population growth fraction, allow one to characterize the rate of cell proliferation that under malignant growth is the determinant of cell proliferative status, which has a great impact on tumor progression, is discussed. The data on the diagnostic value of these proteins as an independent predictor for a broad spectrum of tumors are considered.

Dynamics of replication foci in early S phase as visualized by cross-correlation function.

To monitor gradual changes in the replication foci distribution during early S phase, different segments of newly synthesized DNA were visualized by immunocytochemical mapping of two consecutively incorporated deoxythymidine analogs in pulse-chase-pulse experiments in HeLa cells. The resulting dual-labeled fluorescence images were evaluated using cross-correlation function (CCF) analysis. General changes of CCF shape due to image deterioration caused by blur, noise, and lateral sampling (pixel size) were also discussed. Using CCF analysis of model images simulating either random initiation of new replication foci, or the firing of new foci in close proximity to completed ones, we were able to ascribe the changes in the early S replication foci distribution to the latter mechanism. In contrast to the data published previously, we monitored the dynamics of all replication foci for up to 3 h. In addition, we showed that the replication foci dynamics is well described by random walk model, so that the average de-localization of individual foci is proportional to square root of the applied chase.

Characteristics of cellular proliferation in the developing human retina.

The expression of proliferation-associated proteins Ki67 and PCNA was studied in the retinal rudiments of human embryos at 5-8 weeks of development; studies also addressed the numbers of nucleoli in the nuclei of neuroepithelial cells (with consideration of their distances to the apical surface) and DNA-synthesizing cells after transient (20 min) in vitro incubation in serum-free medium containing BrdU. The retinal rudiment of embryos at five weeks of development had neuroepithelium of the typical structure. BrdU-positive nuclei and nuclei with small numbers of nucleoli were located in the basal part of the ventricular zone. However, this organization was disrupted during the initial period of formation of the inner nuclear layer (six weeks). At this time, DNA-synthesizing cells were found even at the apical surface. Retinal rudiments of embryos at 6-7 weeks of development contained an additional area of cell proliferation in the Chievitz layer and the inner nuclear layer. In eight-week embryos, dividing cells were located in the outer nuclear layer, which again acquired the organization typical of neuroepithelium.

[Characteristics of the cell proliferation in the developing human retina]. / Osobennosti kletochnoi proliferatsii v razvivaiushcheisia kletchatke cheloveka.

The retinal primordia were studied in human embryos at developmental weeks 5-8 to examine the expression of proliferation-associated proteins (Ki67 and PCNA), the number of nucleoli in the nuclei of neuroepithelial cells (in respect to their distance to the apical surface) and the distribution of DNA-synthesizing cells after a short-term (20 min) incubation in vitro in serum-free medium containing BrdU. In 5-week embryos, retinal primordium contains the neuroepithelium with a typical structure. BrdU-positive nuclei and nuclei with small number of nucleoli were found in the basal portion of the ventricular zone. However, this organization was disturbed in the initial period of the formation of inner nuclear layer (week 6). At this stage DNA-synthesizing cells were seen even at the apical surface. In retinal primordia of embryos at weeks 6-7 there appeared an additional area of cell proliferation in the Chievitz layer and in the inner nuclear layer. In embryos at week 8 the dividing cells were concentrated in the outer nuclear layer, which has regained the organization, typical for a neuroepithelium.

[Vascularization and development of the cytoarchitecture in the human neocortical rudiment]. / Vaskuliarizatsiia i stanovlenie tsitoarkhitektoniki v zakladke neokorteksa cheloveka.

The development of cytoarchitectonics of the brain rudiments in mammals is accompanied by the formation of an intracerebral vascular network. The relationship between these two processes is insufficiently clear. We studied the development of blood vessels and cytoarchitectonics in the neocortical rudiment of 6- to 13-week old human embryos. The light and electron microscopy methods were used, as well as histochemical visualization of NADPH-diaphorase in the vessel cells. The endothelium proliferation was evaluated using antibodies to proliferating cell nuclear antigen. Starting from week 8 of development, the tangentially oriented vessels formed a intraneural network in the ventricular zone of the rudiment, which appears to restrict the motility of neuroepithelial cells. The basal membrane was initially absent, and the neuroepithelial cells were in direct contact with the endothelial cells. During week 9 of development, the tangentially oriented vessels appeared in the intermediate zone. Formations similar to glial legs with short regions of the basal membrane adjoined the walls of inter- and intraneural vessels (note that, according to the published data, glial fibrillary acidic protein is not yet visualized at this stage). Angioarchitectonics depended little on the cell population density in different zones of the rudiment; specifically, the cortical plate did not contain tangentially oriented vessels until week 12-13 of development. The data we obtained suggest that the blood vessels fulfill a special morphogenetic function in the developing neocortex.

Numerical simulation of local heat transfer in rotating two-pass square channels.

3D turbulent air flow and heat transfer developing in a two-pass square channel rotating in the orthogonal mode are simulated using the high-Re k-epsilon turbulence model and a recently developed modification of wall functions. Auxiliary problem for accurate definition of inlet boundary conditions formed by a long unheated upstream section is considered. Details of flow structure are presented. Local heat transfer results are compared with experimental data.

[The structure of the developing blood vessels of the neocortical anlage of the human embryo]. / Stroenie formiruiushchikhsia krovenosnykh sosudov zakladki neokorteksa émbriona cheloveka.

Using light and electron microscopy the structure of blood vessels of neocortical anlage of human 7-12 embryos was studied. It was shown that at the early stage of formation of intraorgan vascular network the wall of blood vessels of ventricular zone successively differentiate, which is characterized by the appearance of second layer of cells (pericytes), accumulation of basement membrane components, widening of the zone of contacts between endotheliocytes and establishment of the contacts with bipolar cells of neocortex anlage. The morphological data obtained assist in comprehension of physiological aspects of formation of blood brain barrier and regulation of blood flow in human embryonal neocortex.